Light-activated Cyclases: PAC & Cyclop

Already in 2002, a Japanese working group discovered light-activated adenylyl cyclases in the monocellular flagellate Euglena gracilis (Iseki et al., 2007, Nature) which they named photoactivated adenylyl cyclase (PAC) a and PACb. The use of PAC from Euglena (EuPAC) as an optogenetic tool in animal cells and the fruit fly Drosophila melanogaster (by Martin Schwärzel, now FU Berlin) was shown by us in collaboration with other groups in 2007 (Schröder-Lang et al., Nature Methods) , See fig. 1.

Other PACs were found in genome data from microbes and were characterized by us and others. Particularly attractive is PAC from the bacterium Beggiatoa spec. (bPAC), since it is only 350 amino acids in size and has a high ratio of activity in light to activity in the dark (L / D). By mutagenesis and gene fusion we are currently changing and improving the properties of bPAC.

In 2014, a Brazilian group showed that a rhodopsin fusion with a guanylyl cyclase is expressed in the aquatic fungus Blastocladiella emersonii and is activated by green light (Avelar et al., 2014, Curr. Biol.). We synthesized the DNA and characterized the protein after heterologous expression in oocytes or HEK293 cells. After extensive characterization (Gao et al., 2015, Nat. Commun.), We named the protein Cyclase Opsin or Cyclop, and also showed light-activated inward currents after coexpression of a cGMP-sensitive cation channel, see Fig. 2.

We are currently working on gene fusions of Cyclop and mutations to produce an adenylyl cyclase from the guanylyl cyclase: Cyclop-PAC.

The figure shows that the conductivity oft he same cell can be achieved either by administering IBMX and forskolin during time indicated by bar (increase of cAMP via endogenous enzymes, see B) or by exposure to light during time indicated by bar (activation of PAC, see C).
(Graphic: G. Nagel, Universität Würzburg 2017)